Regarding Malignant Tumor:
A malignant tumor (cancer) is the first leading cause of death in Japan and the number of patients is increasing every year, and the development of a drug and a therapeutic method having high efficacy and safety is strongly desired. Examples of the cause of forming a malignant tumor include a mutation of DNA caused by radiation, ultraviolet rays and various carcinogenic substances. Studies on malignant tumors have been focused on identification of these genetic changes molecular biologically. As a result, it is considered that tumorigenic transformation is induced by accumulation of a large number of mutations and the like. It has been shown by a cell line model and the like that some decisive mutations directly connected with the tumorigenic transformation. Regarding leukemia as one of the objective diseases of the invention, many chromosomal abnormalities have been identified and classified. In many of the case, translocation of chromosome is found and the translocation associated genes have already been identified in the main translocation of chromosome. By functional analyses of the translocation related genes, a case has been found that these genes relates to the onset of leukemia.
Regarding Cancer Stem Cell:
On the other hand, a so-called cancer stem cell hypothesis has been proposed for a long time from the viewpoint of cell biology, stating that stem cell is the origin of a malignant tumor similar to the normal tissue. The stem cell is defined as a cell having self-renewal capacity and pluripotency and generally divided roughly into totipotency stem cell and tissue stem cell. The tissue stem cell is origin of specific tissues and organs such as of blood system, liver, nerve system and the like and present at an extremely low frequency. Among them, the study of hematopoietic stem cell is at the most advanced stage. It has been reported that a hematopoietic system can be reconstituted over a long period of time by transplanting one hematopoietic stem cell into a mouse in which the hematopoietic system was destructed by a lethal dose of irradiation (Non-patent Reference 1). Different from the normal stem cell, studies on cancer stem cells have been delayed for a long time since their true nature could not been found. However, a cancer stem cell has been identified for the first time in acute myelocytic leukemia, in 1997 by Dick et al (Non-patent Reference 2). Thereafter, the presence of cancer stem cells was reported in various malignant tumors. In summing up, cancer stem cells are present at a frequency of several % or less of the whole tumor and the presence of them are rare as well as normal stem cells. It is considered that the remaining cells which form the tumor are tumor precursor cells in which proliferation ability is limited or tumor cells.
From these reports, it was shown that hierarchy is present even in tumor as well as the normal tissue, and the cancer stem cell residing at this top (origin) has strong tumor forming ability. Based on the above, it is considered that the beginning of the onset of malignant tumors is a change from a normal stem cell to a so-called cancer stem cell by addition of several mutations.
Characteristics and Therapeutic Problems of Cancer Stem Cells:
In summing up many reports, it is considered that cancer stem cells are maintaining various characteristics possessed by the normal stem cells. Examples of similarities include rarity of the cell, a microenvironment (niche) in which the stem cells exist, expression of a multiple drug resistance gene, cell cycle, and the like.
Particularly, the characteristics that cancer stem cells express a group of multiple drug resistance genes and are at the stationary phase of cell cycle similar to the normal stem cells could become a therapeutically great problem. A multiple drug resistance gene BCRP is a pump which impairs the drug efficacy by discharging various antitumor agents into outside of cells, and a method for collecting stem cells making use of the activity has been reported (Non-patent Reference 3). In addition, the stem cell is under a state of “hibernation” in order to keep providing cells for its whole life (Non-patent Reference 4) and it reduces in sensitivity for many antitumor agents and radiation (Non-patent References 5 and 6). Based on the above characteristics, it is considered that the rare cancer stem cell which shows resistance to the therapy is a cause of tumor recurrence.
Regarding Molecular Target Drug:
Three main courses of the treatment of a malignant tumor include antitumor agent therapy, radiation therapy and excision. Treatment for the blood tumor is limited to the antitumor agent therapy and radiation therapy, and as described in the above, the cancer stem cell can have a resistance to these treatments. Another problem is that side effects are large since these two treatments affect the entire body. It is a molecular target drug that is expected as a resolving means for this problem. It has a possibility to reduce side effects by exhibiting its drug efficacy only in the cell expressing the target molecule.
Examples of typical drugs of the molecular target drug in the field of blood diseases include imatinib and rituximab. Imatinib targets at a leukemia-causing factor called Bcr-Abl produced by a chromosomal abnormality (Philadelphia chromosome) which is observed in 95% of chronic myeloid leukemia (CML) patients. Imatinib is a low molecular weight drug which induces suicide of leukemia cell by inhibiting function of Bcr-Abl. Rituximab is a therapeutic antibody which recognizes CD20 as a surface molecule on a B cell and has an antitumor effect on non-Hodgkin lymphoma, a malignant tumor of B cell. On the other hand, molecular target drugs for AML are few, and there is only an agent gemtuzumab ozogamicin (Mylotarg) in which an antibiotic calicheamicin is bound to a monoclonal antibody to CD33 known as an AML cell surface antigen (Non-patent Reference 7). However, it can be said that that the use of Mylotarg is limited since Mylotarg can be applied only when a patient meets the following four limitations such as the expression rate of CD33 of 80% or more, a case of recurrency, age of 60 or more, and resistance to other chemical therapy. Based on the above, it can be said that discovery of a new target gene and development of a therapeutic agent for this are important inventions which directly lead to the possibility of therapy and expansion of the choices.
Regarding Embodiment of Molecular Target Drugs:
As the embodiment of molecular target drugs, various substances have been studied and developed such as a therapeutic antibody, a low molecular weight drug, a peptide drug, a biological protein preparation such as cytokine, a siRNA, aptamer and the like. When an antibody is used as a therapeutic agent, due to its specificity, it is useful in treating pathological conditions in which a tumor specific antigen exhibits a property of different cells. The antibody binds to a tumor specific antigen which is a protein expressing on the cell surface and effectively acts upon such cells. The antibody has a characteristic of long blood half life and high specificity for its antigen and is also particularly useful as an antitumor agent. For example, when an antibody targets at a tumor-specific antigen, it can be expected that the administered antibody accumulates into the tumor and thereby attacks the tumor cell via complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) based on the immune system. In addition, by binding a radioactive substance, a cytotoxic substance and the like to an antibody, it becomes possible to transfer an agent efficiently to the tumor part and thereby to allow to act thereon. At the same time, since it can decrease the amount of the reached agent to non-specific other tissues, reduction of side effects can also be expected. Termination or regression of growth of tumor can be expected by activity of antibody accumulated at tumor selectively. Administering antibody will be able to select from antibodies, having agonistic activity when a tumor-specific antigen has an activity to induce cell death, or having neutralization activity when a tumor-specific antigen relates to in the growth and survival of cells. Due to the above characteristics, it is considered that antibodies are suited in applying as antitumor agents.
Regarding Therapeutic Antibodies:
In the original antibody preparation, a mouse was used as the animal to be immunized. However, use of mouse antibodies as drugs in vivo is limited due to a large number of reasons. A mouse antibody which can be recognized as a foreign substance in a human body can induce so-called “human anti-mouse antibody” namely “HAMA” response (Non-patent Reference 8). Further, the Fc region of mouse antibody is not effective for stimulation of human complement or cellular cytotoxicity.
As one of the approaches for avoiding such problems, a chimeric antibody has been developed (EP patent application No. 120694, EP patent application No. 125023). The chimeric antibody contains parts of antibodies derived from two or more species (mouse antibody variable region, human antibody constant region and the like). An advantageous point of such a chimeric antibody is that it keeps the characteristics of a mouse antibody but can stimulate human complement or cellular cytotoxicity since it has human Fc. However, it is known that such a chimeric antibody also induces “human anti-chimeric antibody” namely “HACA” response (Non-patent Reference 9).
Further, it has been developed a recombinant antibody in which only parts of an antibody, complementarity determining regions (“CDR”), were substituted (Patent References 1 and 2). By the use of a CDR grafting technique, an antibody comprising mouse CDR and human variable region framework and constant region, so-called “humanized antibody” (Non-patent Reference 10).
Regarding TIM-3:
TIM gene family comprises eight genes in mice and three genes in humans, and these genes are located at chromosome 11 and at gene region 5q33, respectively (Non-patent Document 11). These gene regions are known to relate to autoimmune diseases and allergic diseases. TIM protein is a type I transmembrane protein having a structurally conserved immunoglobulin variable (IgV) domain and a mucin domain. TIM protein was considered to be specifically expressed on T cells and directly regulate the T cell activity, but there are recent reports on expression of TIM-3 protein in antigen-presenting cells and on their functions (Non-patent Document 12). According to the crystal structure analysis, the TIM protein has a conserved protein structure and has a ligand binding site in the IgV domain.
TIM-3 was identified as a molecule specifically expressed on mouse Th1 cells but not on Th2 cells (Non-patent Document 13). In mice, by binding of TIM-3 to its ligand, galectin9, apoptosis is induced in a mouse Th1 cell, the Th1 response is inhibited, and then to lead to induction of peripheral tolerance. In humans, as similar to mice, TIM-3 is selectively expressed on Th1-cells, as well as phagocytic cells such as macrophages and dendritic cells. It is found that the reduction of expression of human TIM-3 by siRNA or inhibition by a blocking antibody increased the secretion of interferon γ (IFN-γ) from CD4 positive T-cells. This supports the inhibitory role of TIM-3 in human T cells. Analysis of clinical samples from autoimmune disease patients showed no expression of TIM-3 in CD4 positive cells. In particular, expression level of TIM-3 is lower and secretion of IFN-γ is higher in T cell clones derived from the cerebrospinal fluid of patients with multiple sclerosis than those in clones derived from normal healthy persons (Non-patent Document 14).
There are reports on relation of TIM-3 with allergic diseases and/or asthma (Patent Documents 3 and 4). However, the relation between TIM-3 and blood cancer is disclosed only in the report on the microarray analysis of hematopoietic stem cell from acute myeloid leukemia patients and normal hematopoietic stem cells (Non-patent Document 15) and many of the relation between TIM-3 and blood cancer has not been found out.